PROJECT SUMMARY Yellow fever virus (YFV) is the prototype flavivirus and is historically the most important arthropod-borne viral pathogen of humans worldwide with ~200,000 infections annually and a mortality of ~50% in those who develop severe symptoms. YFV is endemic throughout Africa and South America and had been largely controlled through mass vaccination. The YFV vaccine 17D is considered one of the most effective live-attenuated virus (LAV) vaccines ever developed. Even so, every 10-year boosts have been recommended to maintain immunity. However, falling vaccination rates have led to a dramatic resurgence of disease in both Africa and South America, and subsequent vaccination campaigns have depleted the global supply of 17D. In response to these vaccine shortages, the WHO and CDC revised the 10-year boost to a once-in-a-lifetime vaccination recommendation, despite limited supporting data: although serosurveys find that ~90% of vaccinees have detectable neutralizing antibodies to YFV, careful review of these surveys finds that among individuals living in YFV non-endemic settings, at least 20% of YFV vaccinees lack detectable neutralizing antibodies at >10 years post-vaccination. While this finding must be critically evaluated in the context of ongoing outbreaks and vaccine shortages, it also represents a unique opportunity to study how 17D induces and maintains neutralizing antibodies in some vaccinees but not in others. Our central premise is that long-term YFV immunity is established by host immune activation in response to vaccine viremia at the time of vaccination: downstream effects of detectable differences in duration and magnitude of vaccine viremia at vaccination determine whether or not a vaccinee develops life-long immunity. We propose to evaluate this premise and its broader implications in three separate Aims: Aim 1 tests the hypothesis that vaccine viremia correlates with the long-term durability of of YFV neutralizing antibodies. We will enroll YFV pre-vaccinees and prospectively characterize acute vaccine viremia, acute innate immune and adaptive immune responses, and neutralizing antibody titers up to 5 years thereafter. Aim 2 tests the hypothesis that at least 20% of 17D vaccinated subjects will lose YFV immunity between 3- and 7-years post vaccination. We will recruit and prospectively follow a cohort of 17D vaccinees vaccinated 2-3 years prior to enrollment, comparing changes in YFV neutralizing antibodies and other immune markers over time and characterizing individual and cohort antibody decay kinetics. In Aim 3 we use 17D revaccination as a live-virus challenge to test the hypothesis that neutralizing antibody titers correlate with YFV protection. We will prospectively characterize pre-boost antibodies titers, vaccine viremia, acute immune responses and post-boost titers in vaccinees receiving boost 17D vaccinations. We expect to identify neutralizing antibody titers above which sterilizing immunity is conferred and titers below which it is not. These Aims will set a foundation for future studies to further dissect determinants of 17D and other LAV induced immunity and establish metrics that could allow efficient prioritization of 17D vaccination and optimize 17D use in the face of current and future outbreaks.